Exposure to ferrous-like metals lead to multiple toxic endpoints, including lung and erythropoietic abnormalities, and cancer. Many different mechanisms have been proposed to explain the toxicity of ferrous-like metals. Our preliminary data and recent publications suggest that the hypoxia signaling system may play a role in metal induced injury. The ability to sense and cope with low oxygen tension, or hypoxia, is critical to normal physiology and several pathological conditions. Organisms have developed a mechanism of coping with hypoxia and central to this process are the hypoxia inducible factors (HIFs). HIFs are inducible transcription factors that regulate the expression of a battery of genes essential to dealing with a hypoxic environment. These hypoxia responsive genes are also affected by ferrous-like metals, such as cobalt and nickel. Recently, a family of prolyl hydroxylases was identified that act as oxygen sensors for the HIF proteins. These enzymes can be inhibited by ferrous-like metals and offer a link between hypoxia and metal exposure. This intersection has led us to the following hypothesis: HIF mediated transcription is necessary for cellular damage and lung toxicity caused by exposure to ferrous-like metals. To address this hypothesis five specific aims are proposed: 1) Create and characterize ARNT deficient cells. 2) Characterize the role of HIF signaling in metal induced toxicity with HIF deficient cell lines. 3) Determine whether metal induced toxicity is dependent upon stabilization of the HIFs through inhibition of prolyl. hydroxylation. 4) Determine the role of HIF regulated genes in mediating metal induced toxicity using RNAi. 5) Determine whether HIF mediated signaling is necessary for metal induced lung toxicity using HIF1 n and ARNT conditional null mice. The completion of the proposed research will increase our understanding of the signaling mechanism used by ferrous-like metals. The studies will link ferrous-like metals to the hypoxia response pathway and its down-stream target genes. The proposal will create a set of cell lines that will have broad interest in several fields, including oncology, toxicology, and pharmacology. In addition, the understanding we gain about hypoxia and HIF mediated signaling will have benefits to developmental and cancer biology. Finally, these studies will elucidate the role of hypoxia signaling in metal induced toxicity in vitro and in vivo.